This grant application aims to understand the gene pathways that pattern the retina and control guidance receptor expression to establish the crossed and uncrossed visual projections. Our previous work defined such a program for the uncrossed or ipsilateral projection: the guidance receptor EphB1 is expressed in ventrotemporal (VT) retinal ganglion cells (RGCs), and repulsive interactions between EphB1-expressing VT RGCs and EphrinB2-expressing radial glia at the optic chiasm midline establish the ipsilateral projection. The transcription factor Zic2 controls EphB1 expression, is necessary and sufficient for inducing an ipsilateral projection, and regulates factors important for activity-dependent target innervation. We have recently identified a contralateral RGC midline guidance program: the Ig-CAM Nr-CAM and the semaphorin receptor Plexin-A1, expressed by contralateral RGCs and optic chiasm cells, form a complex with midline Sema6D to convert inhibition into growth of contralateral RGC axons. However, little is known about the transcriptional control of the contralateral RGC projection. The proposed studies seek to uncover transcriptional networks controlling ipsilateral and contralateral RGC identity and retinal patterning. Aim 1 will analyze transcriptional pathways of the contralateral projection. We have discovered that the SoxC group of transcription factors (Sox4, 11 and 12) is expressed in crossed but not uncrossed RGCs and binds to the promoter regions of Plexin-A1 and Nr-CAM. Using a mouse model conditionally removing all three SoxCs, we will determine whether SoxCs regulate the contralateral projection through directing contralateral guidance molecule expression. We will also determine whether Zic2 represses contralateral genes to maintain uncrossed RGC identity. In Aim 2, we will further investigate how crossed and uncrossed projections are specified by focusing on transcription factors upstream of those studied in Aim 1, namely, Foxg1 and Foxd1. Foxd1 is expressed in the VT retina and is required for Zic2 and EphB1 expression, thus placing it upstream of the transcriptional program for the ipsilateral projection. As Foxg1 is expressed in the areas giving rise to contralaterally projecting RGCs, we will investigate whether Foxg1 controls the contralateral projection through regulation of contralateral gene expression, using a novel conditional Foxg1 mouse. In Aim 3, we build on our recent success in gene expression profiling to identify genes that are differentially expressed in ipsilateral versus contralateral RGCs. We will characterize their expression patterns and roles in directing the formation of the binocular circuit. Together, these studies will illuminate how the ipsilateral and contralateral retinal sectors are specified - information that is essential for implementing regeneration of indigenous RGCs or their stem cell replacements. PUBLIC HEALTH RELEVANCE: This research aims to understand the regulation of cell identity of retinal ganglion cells that grow out from each eye, meet at the X-shaped optic chiasm, then diverge toward targets on the same and opposite side of the brain. Proper binocular vision is dependent on a normal distribution of retinal axons crossing at the optic chiasm, and if altered, reduced visual acuity and depth perception ensue. This work investigates the gene networks that control identity and partitioning of the retinal ganglion cells that project ipsi- an contralaterally to enable axonal navigation through the appropriate route.